Preventives/remedies for proliferative organ diseases chronic arthritic diseases. hypertrophic scar or keloid

ABSTRACT

An agent for prophylaxis or treatment of proliferative organ diseases, chronic arthritic diseases, hypertrophic scars or keloid, comprising an erythropoietin receptor antagonist or a salt thereof.

TECHNICAL FIELD

[0001] The present invention relates to an agent for prophylaxis ortreatment of proliferative organ diseases, chronic arthritic diseases,and hypertrophic scars or keloid, comprising an erythropoietin receptorantagonist, etc.

BACKGROUND ART

[0002] As a therapeutic method for proliferative organ diseases such ascancer and proliferative lesion in tissues or organs, surgicaldissection, irradiation, administration of anticancer agents orcombination thereof has been conventionally used. However, basic andspecific investigations for the biological characteristics of canceritself fall behind the remarkable progress of diagnosis technologies forcancer. Therefore, under the present circumstances, drastic therapy ofcancer has not been established yet.

[0003] Erythropoietin is involved in proliferation and differentiationof hematocytes. Unlike other cytokines, erythropoietin is not producedin hematocyte but produced in kidneys or liver and released into blood.Erythropoietin is considered to act on erythroid burst forming cell(BFU-E) and erythroid colony forming cell (CFU-E) among erythroidprecursor cells, stimulate their proliferation and differentiation, andinduce production of erythrocytes (Krantz S. B., Blood, Vol. 77, pp.419-434 (1991)). It has been considered that when erythropoietin bindsto a erythropoietin receptor existing on the cell membrane of aprecursor cell, a signal is transmitted into the cell nucleus to causedifferentiation into an erythrocyte, i.e., accumulation of globin mRNAin the cell, production of hemoglobin and differentiation into anerythrocyte (D'Andrea A. D. et al., Cell, Vol. 57, pp. 277-285 (1989)).However, the detailed mechanism thereof has not yet been clarified, andmany problems still remain to be solved.

[0004] As the sites on which erythropoietin expresses its gene amongtissues except the sites relating to erythroblast, embryos of the earlypost-implantation stage (Yasuda Y. et al., Develop. Growth Differ., Vol.35, pp. 711-722 (1993)), brains of humans, monkeys and mice (Marti H. H.et al., Eur. J. Neu. Sci., Vol. 8, pp. 666-676 (1996)) and endometria ofmice (Yasuda Y. et al., J. Biol. Chem., Vol. 273, pp. 25381-25387(1998)) are known. Furthermore, the present inventors have been foundthat the erythropoietin receptor gene is expressed on mouse deciduae(Yasuda Y. et al., Develop. Growth Differ., Vol. 35, pp. 711-722 (1993))and vascular endothelium cells of mouse endometrium (Yasuda Y. et al.,J. Biol. Chem., Vol. 273, pp. 25381-25387 (1998)) in addition toerythroblasts. Under the present circumstance, the functions oferythropoietin or erythropoietin receptor genes on such sites other thanhematocytes have not been revealed yet.

[0005] When an embryo nidates on an endometrium, the implantation siteof the endometrium undergoes decidual reaction, whereby the deciduaesurround the embryo. The erythropoietin receptor gene is expressed ondeciduae, whereas erythropoietin is not expressed thereon. Accordingly,it is considered that the erythropoietin receptor is produced ondeciduae and bound to the erythropoietin in blood to transmit theerythropoietin signal. According to the investigation on normal humanendometrium, expression of the erythropoietin gene could be observed insome samples and could not be observed in other samples by the presenttechnology. However, expressions of erythropoietin and an erythropoietinreceptor at the level of protein were observed in all samples.Accordingly, it is considered that erythropoietin is taken from blood orself-secreted at the extremely low concentration in the human normalendometrium, which is similar to the case of the deciduae, and involvedin normal physiological function of uterus. On the other hand, it hasbeen recognized by RT-PCR and Southern Blot method that theerythropoietin mRNA is expressed on cervical cancer, corpus utericancer, hysteromyoma, ovarian cancer, and ovarian cystoma. Furthermore,an erythropoietin receptor is expressed on the vascular endotheliumcells in these cancer tissues. The investigation on these tissues hasrevealed that erythropoietin and erythropoietin receptor proteins, aswell as proliferative nuclear antigens exist on these cancer cells.Accordingly, it was presumed that erythropoietin is involved inproliferation of cancer cells.

[0006] Furthermore, it is disclosed that a substance that binds to anerythropoietin receptor in a specific domain can be used for therapy ofchronic rheumatoid arthritis (WO00/66632).

[0007] Along the way, the present inventors have found thaterythropoietin antagonists (the “erythropoietin antagonist” means asubstance capable of binding to erythropoietin) such as erythropoietinantibodies, erythropoietin receptor proteins, etc., have an inhibitoryeffect on proliferation of cancer cells and an interrupting effect onintervening blood capillaries (JP-A-10-101574, British Journal ofCancer, Vol. 84, pp. 836-843 (2001)).

OBJECT OF THE INVENTION

[0008] However, all of these erythropoietin antagonists are proteins,and any low molecular weight compounds such as peptides having both aninhibitory effect on proliferation of cancer cells and an effect onvascular proliferation in cancer tissues by the similar action mechanism(blocking of the erythropoietin signal) have not been known yet. Thepresent invention aims at providing a superior agent for prophylaxis ortreatment of proliferative organ diseases, chronic arthritic diseases,and hypertrophic scars or keloid, etc., which comprises a low molecularweight compound such as peptide as an active component.

SUMMARY OF THE INVENTION

[0009] The present inventors have done intensive investigations to solvethe above-mentioned problem, and found that a low molecular weightcompound such as a peptide having erythropoietin receptor antagonism hasunexpectedly superior inhibitory effects on proliferation of both acancer cell and an intercalated blood vessel. Based on these findings,the present inventors have done further investigations, which resultedin completion of the present invention.

[0010] Namely, the present invention relates to:

[0011] [1] an agent for prophylaxis or treatment of proliferative organdiseases, chronic arthritic diseases, hypertrophic scars or keloid,comprising an erythropoietin receptor antagonist or a salt thereof;

[0012] [2] the agent according to the above-mentioned [1], wherein theerythropoietin receptor antagonist or a salt thereof is a low molecularweight erythropoietin receptor antagonist or a salt thereof;

[0013] [3] the agent according to the above-mentioned [1], wherein theerythropoietin receptor antagonist or a salt thereof is a low molecularweight peptidic erythropoietin receptor antagonist or a salt thereof;

[0014] [4] the agent according to the above-mentioned [1], wherein theerythropoietin receptor antagonist or a salt thereof is anerythropoietin mimetic peptide or a salt thereof;

[0015] [5] the agent according to the above-mentioned [1], wherein theerythropoietin receptor antagonist or a salt thereof is erythropoietinmimetic peptide 9 or a salt thereof;

[0016] [6] the agent according to the above-mentioned [1], wherein theerythropoietin receptor antagonist or a salt thereof is a peptidecomprising an amino acid sequence identical or substantially identicalwith the amino acid sequence of SEQ ID NO: 1 or a salt thereof;

[0017] [7] the agent according to the above-mentioned [1], wherein theerythropoietin receptor antagonist or a salt thereof is ananti-erythropoietin receptor antibody or a salt thereof;

[0018] [8] the agent according to the above-mentioned [1], wherein theproliferative organ disease is cancer or tumor;

[0019] [9] the agent according to the above-mentioned [1], wherein thechronic arthritic disease is rheumatoid arthritis, rheumatoid diseases,chronic arthritis of collagenosis or tendovaginitis;

[0020] [10] a vascular proliferation suppressing agent comprising anerythropoietin receptor antagonist or a salt thereof;

[0021] [11] a method for prophylaxis or treatment of proliferative organdiseases, chronic arthritic diseases, hypertrophic scars or keloid,comprising administering an effective amount of an erythropoietinreceptor antagonist or a salt thereof to a mammal;

[0022] [12] a method for prophylaxis or treatment of proliferative organdiseases, comprising administering an effective amount of anerythropoietin receptor antagonist or a salt thereof in combination withan effective amount of another anticancer agent or a salt thereof to amammal;

[0023] [13] use of an erythropoietin receptor antagonist or a saltthereof for production of an agent for prophylaxis or treatment ofproliferative organ diseases, chronic arthritic diseases, hypertrophicscars or keloid, etc.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The erythropoietin receptor antagonist used for the agent forprophylaxis or treatment of proliferative organ diseases, chronicarthritic diseases, hypertrophic scars or keloid of the presentinvention (hereinafter abbreviated to as “the agent of the presentinvention”) is not specifically limited as long as it is a substancethat blocks the signaling from an erythropoietin receptor bycompetitively or non-competitively inhibiting binding of erythropoietinto the human erythropoietin receptor, or a substance that blocks thesignaling from an erythropoietin receptor without inhibiting binding oferythropoietin to the human erythropoietin receptor (e.g., a substancethat inhibits JAK2 kinase, etc.). For example, anti-erythropoietinreceptor antibodies, etc. are included.

[0025] Among these, preferred erythropoietin receptor antagonist is alow molecular weight compound having a molecular weight of about 200 to5000 and specifically a low molecular weight compound having a molecularweight of about 300 to 3000. Although the erythropoietin receptorantagonist may be peptidic or non-peptidic, it is preferably peptidicand more preferably peptidic one having a low molecular weight.

[0026] The low molecular peptide used for the present inventionincludes, for example, a peptide consisting of at least 5 or more,preferably about 5 to 30, specifically preferably about 15 to 25 ofamino acid residues, etc.

[0027] The erythropoietin mimetic peptide (EMP) used for the presentinvention is not specifically limited as long as it is a peptide thatblocks the signaling from an erythropoietin receptor by binding to thehuman erythropoietin receptor with the strength of binding between aligand and a receptor, or a peptide that blocks the signaling from anerythropoietin receptor without binding to the human erythropoietinreceptor (e.g., a peptide that inhibits JAK2 kinase, etc.). For example,erythropoietin mimetic peptides (EMPs) described in Biochemistry 37,3699, 1998 are included. Among these, EMP6, EMP7, EMP9, EMP12, EMP22,EMP23, EMP24, EMP25, EMP33 and EMP39 are preferred, and EMP9 isspecifically preferred.

[0028] More specifically, for example, a peptide comprising an aminoacid sequence identical or substantially identical with the amino acidsequence represented by SEQ ID NO: 1, etc. is used as the erythropoietinreceptor antagonist. The peptide may be any of peptides, syntheticpeptides and recombinant peptides derived from any cells of mammals(e.g., guinea pig, rat, mouse, chicken, rabbit, swine, sheep, bovine,monkey, human, etc.), for example, hepatic cells, spleen cells, nervecells, glia cells, β-cells of pancreas, bone marrow cells, mesangialcells, Langerhans' cells, epidermal cells, epithelial cells, endothelialcells, fibroblasts, fibrocytes, striated muscle cells, smooth musclecells, fat cells, immunocytes (e.g., macrophage, T cells, B cells,natural killer cells, mast cells, neutrophils, basophils, eosinophils,monocytes, etc.), megakaryocytes, synovial cells, chondrocytes,osteocytes, osteoblasts, osteoclasts, mammary gland cells orinterstitial cells, or precursor cells, stem cells or cancerous cellsthereof, etc., or any tissues containing such cells, for example, brain,various parts of brain (e.g., olfactory bulb, amygdala, cerebral basalganglia, hippocampus, thalamus, hypothalamus, diencephalic cerebralcortex, medulla oblongata, cerebellum), spinal cord, pituitary, stomach,pancreas, kidney, liver, genital gland, thyroid gland, gallbladder, bonemarrow, adrenal gland, skin, muscle, lung, digestive tract (e.g., largeintestine, small intestine), blood vessel, heart, thymus, spleen,sublingual gland, parotid gland, peripheral blood, prostate, testis,ovary, placenta, uterus, bone, joint, skeletal muscle and connectivetissues of these tissues, etc., or hematocyte cells or cultured cellstrains thereof, etc.

[0029] The peptide comprising an amino acid sequence substantiallyidentical with the amino acid sequence represented by SEQ ID NO: 1includes a peptide comprising an amino acid sequence having at leastabout 70% homology, preferably at least about 80% homology, morepreferably at least about 90% homology, most preferably at least about95% homology, to the amino acid sequence represented by SEQ ID NO: 1,etc.

[0030] Furthermore, as the erythropoietin receptor antagonist, forexample, a peptide comprising an amino acid sequence in which 1 or 2 ormore (preferably about 1 to 7, more preferably about 1 to 5, morepreferably 1 to 3) of amino acids have been deleted from the amino acidsequence represented by SEQ ID NO: 1; a peptide comprising an amino acidsequence in which 1 or 2 or more (preferably about 1 to 7, morepreferably about 1 to 5, more preferably 1 to 3) of amino acids havebeen added to or inserted in the amino acid sequence represented by SEQID NO: 1; a peptide comprising an amino acid sequence in which 1 or 2 ormore (preferably about 1 to 7, more preferably about 1 to 5, morepreferably 1 to 3) of amino acids have been replaced with other aminoacids in the amino acid sequence represented by SEQ ID NO: 1; or apeptide comprising a combination of the above-mentioned amino acidsequences, etc. can be used.

[0031] The other amino acids as used herein may be either natural typeor non-natural type.

[0032] The erythropoietin receptor antagonist is designated by theconventional way of describing peptides. That is, the left end is theN-terminal (amino terminal) and the right end is the C-terminal(carboxyl terminal). In the erythropoietin receptor antagonist includinga low molecular peptide having the amino acid sequence represented bySEQ ID No: 1, the C-terminal is normally a carboxyl group (—COOH) orcarboxylate (—COO⁻), but the C-terminal may be an amide (—CONH₂) or anester (—COOR).

[0033] Examples of the ester group shown by R include a C₁₋₆ alkyl groupsuch as methyl, ethyl, n-propyl, isopropyl or n-butyl; a C₃₋₈ cycloalkylgroup such as cyclopentyl or cyclohexyl; a C₆₋₁₂ aryl group such asphenyl or α-naphthyl; a C₆₋₁₂ aryl-C₁₋₂ alkyl group such as benzyl,phenethyl or α-naphthylmethyl, etc. In addition, pivaloyloxymethylester, etc. that is used generally as an ester for oral administrationmay also be used.

[0034] In the case wherein the erythropoietin receptor antagonistcontains a carboxyl group (or carboxylate) at a position other than theC-terminal, the carboxyl group may be amidated or esterified, and suchamide or ester is also included within the low molecular peptide of thepresent invention. In this case, the ester group may be the same groupas that described with respect to the above C-terminal.

[0035] Furthermore, the erythropoietin receptor antagonist includesderivatives wherein a group (e.g., OH, COOH, NH₂, SH, etc.) on the sidechains of an amino acid in the molecule is protected with a suitableprotecting group (e.g., a C₁₋₆ acyl group such as a formyl group, anacetyl group, etc.), or conjugated peptides such as glycopeptides withsugar chains attached thereto.

[0036] As a specific example of the erythropoietin receptor antagonist,erythropoietin mimetic peptide 9 (EMP9) consisting of the amino acidsequence represented by SEQ ID NO: 1 is preferably used.

[0037] A salt of the erythropoietin receptor antagonist is preferably inthe form of physiologically acceptable acid addition salts. Examples ofsuch salts include salts with inorganic acids (e.g., hydrochloric acid,phosphoric acid, hydrobromic acid, sulfuric acid), salts with organicacids (e.g., acetic acid, formic acid, propionic acid, fumaric acid,maleic acid, succinic acid, tartaric acid, citric acid, malic acid,oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid,aspartic acid, glutamic acid, etc.), etc. Salts with inorganic bases(e.g., alkali metal such as sodium, potassium, etc., alkaline earthmetal such as calcium, magnesium, etc., aluminum or ammonium, etc.),salts with organic bases (e.g., trimethylamine, triethylamine, pyridine,picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine,cyclohexylamine, dicyclohexylamine, N,N′-dibenzylethylenediamine,lysine, arginine, histidine, etc.), etc. can be also used.

[0038] A prodrug of the erythropoietin receptor antagonist or a saltthereof to be contained in the agent of the present invention refers toa compound that converts to an erythropoietin receptor antagonist byreaction of an enzyme, gastric acid, etc. under physiological conditionsin vivo, i.e., a compound that undergoes enzymatic oxidation, reductionor hydrolysis, etc. and thereby converts to an erythropoietin receptorantagonist, or a compound that undergoes hydrolysis, etc. by gastricacid, etc. and thereby converts to an erythropoietin receptorantagonist.

[0039] Examples of the prodrugs of the erythropoietin receptorantagonist or a salt thereof to be contained in the agent of the presentinvention include compounds derived by acylation, alkylation orphosphorylation of the amino group of the low molecular peptide to becontained in the agent of the present invention (e.g., compounds derivedby eicosanoylation, alanylation, pentylaminocarbonylation,(5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxycarbonylation,tetrahydrofuranylation, pyrrolidylmethylation, pivaloyloxymethylation ortert-butylation of the amino group of the low molecular peptide to becontained in the agent of the present invention); compounds derived byacylation, alkylation, phosphorylation or boration of the hydroxyl groupof the low molecular peptide to be contained in the agent of the presentinvention (e.g., compounds derived by acetylation, palmitoylation,propanoylation, pivaloylation, succinylation, fumarylation, alanylationor dimethylaminomethylcarbonylation of the hydroxyl group of the lowmolecular peptide to be contained in the agent of the presentinvention); and compounds derived by esterification or amidation of thecarboxyl group of the low molecular peptide to be contained in the agentof the present invention (e.g., compounds derived by ethylesterification, phenyl esterification, carboxymethyl esterification,dimethylaminomethyl esterification, pivaloyloxymethyl esterification,ethoxycarbonyloxyethyl esterification, phthalidyl esterification,(5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl esterification,cyclohexyloxycarbonylethyl esterification, or methylamidation of thecarboxyl group of the low molecular peptide to be contained in the agentof the present invention), etc. These compounds can be produced from thelow molecular peptide to be contained in the agent of the presentinvention by per se known methods.

[0040] The prodrug of the erythropoietin receptor antagonist or a saltthereof to be contained in the agent of the present invention may be acompound that converts to an erythropoietin receptor antagonist underphysiological conditions as described in “Iyakuhin No Kaihatsu(Development of Drugs)”, Vol. 7, Molecular Designing, published byHirokawa Shoten, 1990, pages 163-198.

[0041] The erythropoietin receptor antagonist or a salt thereof to becontained in the agent of the present invention can be prepared by (1) amethod known per se from the above-mentioned cells or tissues ofmammals, (2) a peptide synthesis method, or (3) a method comprisingculturing a transformant comprising DNA encoding a low molecular peptideto be contained in the agent of the present invention.

[0042] [Preparation Method from Mammalian Cells or Tissues]

[0043] When the erythropoietin receptor antagonist or a salt thereof ismanufactured from human or mammalian tissues or cells, the human ormammalian tissues or cells are homogenized, and then extracted with anacid, etc. The extract can be isolated and purified by a combination ofchromatography techniques such as reverse phase chromatography, ionexchange chromatography, etc.

[Preparation Method According to a Peptide Synthesis Method]

[0044] The erythropoietin receptor antagonist or a salt or an amidethereof to be contained in the agent of the present invention can bemanufactured by a method for peptide synthesis known per se, or bycleaving erythropoietin with a suitable peptidase.

[0045] The peptide synthesis method may be, for example, a solid phasesynthesis method or a liquid phase synthesis method. That is, a partialpeptide or amino acids that can constitute the low molecular peptide tobe contained in the agent of the present invention is condensed with theremaining part of the low molecular peptide and, if the product has aprotecting group, it is removed to obtain the desired peptide. Examplesof known methods for condensation and elimination of a protecting groupare described in the following 1)-5).

[0046] 1) M. Bodanszky & M. A. Ondetti: Peptide Synthesis, IntersciencePublishers, New York (1966)

[0047] 2) Schroeder & Luebke: The Peptide, Academic Press, New York(1965)

[0048] 3) Nobuo Izumiya, et al.: Peptide Gosei-no-Kiso to Jikken (Basicsand experiments of peptide synthesis), published by Maruzen Co. (1975)

[0049] 4) Haruaki Yajima & Shunpei Sakakibara: Seikagaku Jikken Koza(Biochemical Experiment) 1, Tanpakushitsu no Kagaku (Chemistry ofProteins) IV, 205 (1977)

[0050] 5) Haruaki Yajima ed.: Zoku Iyakuhin no Kaihatsu (A sequel toDevelopment of Pharmaceuticals), Vol. 14, Peptide Synthesis, publishedby Hirokawa Shoten

[0051] More specifically, in order to synthesize the erythropoietinreceptor antagonist or a salt or an amide form thereof to be containedin the agent of the present invention, commercially available resins forpeptide synthesis can be used. Examples of such resins includechloromethyl resin, hydroxymethyl resin, benzhydrylamine resin,aminomethyl resin, 4-benzyloxybenzyl alcohol resin,4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylphenylacetamidomethyl resin, polyacrylamide resin,4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)phenoxy resin, etc. Using suchresins, amino acids in which α-amino groups and functional groups on theside chains are appropriately protected are condensed on the resin inthe order of the sequence of the desired low molecular peptide accordingto various condensation methods known per se. At the end of thereaction, the low molecular peptide is excised from the resin and at thesame time, the protecting groups are removed. Then, intramoleculardisulfide bond-forming reaction is performed in a highly dilutedsolution to obtain the desired low molecular peptide or an amide formthereof.

[0052] For condensation of the protected amino acids described above, avariety of activation reagents for peptide synthesis can be used, butcarbodiimides are particularly preferably employed. Examples of suchcarbodiimides include DCC, N,N′-diisopropylcarbodiimide, andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. For activation by thesereagents, the protected amino acids in combination with a racemizationinhibiting additive (e.g., HOBt, HOOBt) are added directly to the resin,or the protected amino acids are previously activated in the form ofsymmetric acid anhydrides, HOBt esters or HOOBt esters, followed byadding the thus activated protected amino acids to the resin.

[0053] Solvents suitable for use in the activation of the protectedamino acids or the condensation with the resin may be appropriatelyselected from solvents that are known to be usable for peptidecondensation reactions. Examples of such solvents include acid amidessuch as N,N-dimethylformamide, N,N-dimethylacetamide andN-methylpyrrolidone; halogenated hydrocarbons such as methylene chlorideand chloroform; alcohols such as trifluoroethanol; sulfoxides such asdimethylsufoxide; ethers such as pyridine, dioxane and tetrahydrofuran;nitrites such as acetonitrile and propionitrile; esters such as methylacetate and ethyl acetate; and appropriate mixtures of these solvents.

[0054] The reaction temperature is appropriately selected from the rangeknown to be applicable to peptide bond-forming reactions and isgenerally selected in the range of about −20°0 C. to about 50° C. Theactivated amino acid derivatives are used generally in an excess amountof 1.5 to 4 times. As a result of a test using the ninhydrin reaction,it was found that when the condensation is insufficient, thecondensation can be completed by repeating the condensation reactionwithout removing of the protecting groups. When the condensation is yetinsufficient even after repeating the reaction, unreacted amino acidsare acetylated with acetic anhydride or acetylimidazole.

[0055] Examples of protecting groups used to protect the amino group ofthe starting material include Z, Boc, tert-pentyloxycarbonyl,isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z,adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl,2-nitrophenylsulphenyl, diphenylphosphinothioyl, Fmoc, etc.

[0056] The carboxyl group can be protected by, e.g., alkylesterification (e.g., esterification of linear, branched or cyclic alkylsuch as methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl), aralkylesterification (e.g., benzyl esterification, 4-nitrobenzylesterification, 4-methoxybenzyl esterification, 4-chlorobenzylesterification, benzhydryl esterification), phenacyl esterification,benzyloxycarbonyl hydrazidation, tert-butoxycarbonyl hydrazidation,trityl hydrazidation, etc.

[0057] The hydroxyl group of serine can be protected by, for example,esterification or etherification. Examples of groups suitably used forthe esterification include a lower alkanoyl group such as an acetylgroup, an aroyl group such as a benzoyl group, a group derived fromcarbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonylgroup, etc. Examples of a group suitably used for the etherificationinclude a benzyl group, a tetrahydropyranyl group, a tert-butyl group,etc.

[0058] Examples of protecting groups for the phenolic hydroxyl group oftyrosine include Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, tert-butyl, etc.

[0059] Examples of protecting groups for the imidazole moiety ofhistidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP,benzyloxymethyl, Bum, Boc, Trt, Fmoc, etc.

[0060] Examples of the activated carboxyl groups in the starting aminoacids include the corresponding acid anhydrides, azides, activatedesters (esters with alcohols (e.g., pentachlorophenol,2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol,p-nitrophenol, HONB, N-hydroxysuccimide, HOBt)), etc. Examples of theactivated amino groups in the starting amino acids include thecorresponding phosphoric amides.

[0061] Methods of eliminating (detaching) the protecting groups includeacid treatment with anhydrous hydrogen fluoride, methanesulfonic acid,trifluoromethanesulfonic acid or trifluoroacetic acid, or a mixturesolution of these acids; base treatment with diisopropylethylamine,triethylamine, piperidine, or piperazine; and reduction with sodium inliquid ammonia. The elimination reaction by the acid treatment describedabove is generally carried out at a temperature of about −20° C. toabout 40° C. In the acid treatment, it is efficient to add a cationscavenger such as anisole, phenol, thioanisole, m-cresol, p-cresol,dimethylsulfide, 1,4-butanedithiol or 1,2-ethanedithiol. Furthermore, a2,4-dinitrophenyl group known as a protecting group for the imidazole ofhistidine is removed by treatment with thiophenol. A formyl group usedas a protecting group for the indole of tryptophan is eliminated by theabove-mentioned acid treatment in the presence of 1,2-ethanedithiol or1,4-butanedithiol, as well as by treatment with an alkali such as adilute sodium hydroxide solution and dilute ammonia.

[0062] Protection of functional groups that should not be involved inthe reaction of the starting material, the protecting groups,elimination of the protecting groups and activation of functional groupsthat are involved in the reaction, etc. may be appropriately selectedfrom known groups and known means.

[0063] In another method for obtaining the object amidated low molecularpeptide, for example, the a-carboxyl group of the carboxy terminal aminoacid is first protected by amidation, and the peptide chain is thenextended from the amino group side up to the desired length. Thereafter,a partial peptide in which only the protecting group of the N-terminalα-amino group of the peptide chain has been eliminated and a partialpeptide in which only the protecting group of the C-terminal carboxylgroup has been eliminated are produced. The two partial peptides arecondensed in a solvent mixture described above. The details of thecondensation reaction are the same as described above. After theprotected peptide obtained by the condensation is purified, and all theprotecting groups are then eliminated by the method described above togive the desired crude low molecular peptide. This crude low molecularpeptide can be purified by various known purification means.Lyophilization of the major fraction gives the amide form of the desiredlow molecular peptide.

[0064] To prepare the esterified form of the low molecular peptide, forexample, the α-carboxyl group of the carboxy terminal amino acid iscondensed with the desired alcohol to prepare the amino acid ester,which is subjected to a procedure similar to the preparation of theamidated low molecular peptide described above to give the desiredesterified low molecular peptide.

[0065] After the reaction, the product may be isolated and purified by acombination of conventional purification methods such as solventextraction, distillation, column chromatography, liquid chromatographyand recrystallization to give the objective peptide. When the lowmolecular peptide obtained by the above methods is in a free form, thepeptide can be converted into a suitable salt by a known method. Whenthe peptide is obtained in a salt form, it can be converted into a freeform by a known method.

[0066] More specifically, the erythropoietin receptor antagonist or asalt thereof to be contained in the agent of the present invention isprepared by a method described in Biochemistry 37, 3699, 1998.

[0067] The erythropoietin receptor antagonist or a salt thereof to becontained in the agent of the present invention may be ananti-erythropoietin receptor antibody or a salt thereof.

[0068] The antibody against an erythropoietin receptor or a salt thereofmay be either a polyclonal antibody or a monoclonal antibody as long asit can recognize an erythropoietin receptor or a salt thereof.

[0069] The antibody against an erythropoietin receptor or a salt thereof(hereinafter sometimes referred to as an erythropoietin receptor, etc.)can be prepared according to a known method for preparing an antibody oran antiserum using an erythropoietin receptor as the antigen.

[0070] [Preparation of Monoclonal Antibody]

[0071] (a) Preparation of Monoclonal Antibody Producer Cells

[0072] An erythropoietin receptor or the like is administered alone orin combination with a carrier or a diluent to a mammal at a site thatcan produce the antibody. In order to enhance the antibody productivity,Freund's complete adjuvant or Freund's incomplete adjuvant may beadministered. The administration is effected usually once every 2 to 6weeks and about 2 to 10 times in total. Mammals to be used includemonkeys, rabbits, dogs, guinea pigs, mice, rats, sheeps, and goats.Preferably, mice and rats are used.

[0073] For preparing monoclonal antibody producer cells, an individualthat shows an antibody titer is selected from warm-blooded animals,e.g., mice, immunized with the antigen, and then the spleen or lymphnode is removed after 2 to 5 days of the final immunization. Monoclonalantibody producer hybridoma cells can be prepared by fusion of theantibody producer cells contained in the spleen or lymph node withmyeloma cells. The antibody titer in the antiserum can be determined,for example, by reacting a labeled erythropoietin receptor as describedbelow or the like with the antiserum and then measuring the activity ofthe labeling agent bound to the antibody. The fusion can be carried outaccording to any known method, for example, the method of Kohler andMilstein (Nature, 256, 495, 1975). Examples of a fusion accelerator arepolyethylene glycol (PEG), Sendai virus, etc. Preferably, PEG is used.

[0074] Examples of myeloma cells include NS-1, P3U1, SP2/0, etc.Preferably, P3U1 is used. The ratio of the number of the antibodyproducer cells (spleen cells) to the number of myeloma cells to be usedis preferably about 1:1 to about 20:1. The cell fusion may be carriedout efficiently by adding PEG (preferably PEG 1000 to PEG 6000) at aconcentration of about 10% to about 80%, followed by incubation at about20° C. to about 40° C., preferably about 30° C. to about 37° C. forabout 1 minute to about 10 minutes.

[0075] For screening monoclonal antibody producer hybridomas, variousmethods can be used. Examples of such methods include a method whichcomprises adding a culture supernatant of hybridoma to a solid phase(e.g., microplate) on which an antigen such as an erythropoietinreceptor is adsorbed directly or together with a carrier, adding ananti-immunoglobulin antibody (where mouse cells are used for the cellfusion, anti-mouse immunoglobulin antibody is used) labeled with aradioactive substance, an enzyme or the like, or Protein A, and thendetecting a monoclonal antibody bound to the solid phase; and a methodwhich comprises adding a culture supernatant of hybridoma to a solidphase on which an anti-immunoglobulin antibody or Protein A is adsorbed,adding an erythropoietin receptor or the like labeled with a radioactivesubstance or an enzyme and then detecting a monoclonal antibody bound tothe solid phase.

[0076] Monoclonal antibodies can be selected according to known methodsor similar methods thereto. In general, the selection can be effected ina medium for animal cells supplemented with HAT (hypoxanthine,aminopterin and thymidine). Any media in which hybridoma cells can growmay be used for screening and growing. For example, RPMI 1640 mediumcontaining 1% to 20%, preferably 10% to 20% fetal bovine serum, GITmedium containing 1% to 10% fetal bovine serum (Wako Pure ChemicalIndustries, Ltd.), a serum free medium for culturing hybridomas(SFM-101, Nissui Pharmaceutical Co., Ltd.), etc. can be used. Theculture temperature is usually 20° C. to 40° C., preferably about 37° C.The culture period is usually about 5 days to about 3 weeks, preferably1 to 2 weeks. The culture may be usually carried out in 5% CO₂. Theantibody titer of the culture supernatant of a hybridoma can bedetermined as in the determination of antibody titer in antiseradescribed above.

[0077] (b) Purification of Monoclonal Antibody

[0078] Isolation and purification of a monoclonal antibody can becarried out according the same manner as applied to conventionalisolation and purification of polyclonal antibodies, for example, amethod of isolation and purification of immunoglobulins (for example,salting-out, alcohol precipitation, isoelectric point precipitation,electrophoresis, adsorption and desorption with ion exchangers (e.g.,DEAE), ultracentrifugation, gel filtration, or a specific purificationmethod in which an antigen-binding solid phase or an activated adsorbentsuch as Protein A or Protein G is used to collect only an antibody, andthe binding is dissociated to obtain the antibody).

[0079] [Preparation of Polyclonal Antibody]

[0080] The polyclonal antibody of the present invention can be producedby known methods or similar methods thereto. For example, the polyclonalantibody can be produced by forming a complex between an immune antigen(an antigen such as a erythropoietin receptor, etc.) and a carrierprotein, immunizing a mammal with the complex in a manner similar to themethod described above for a monoclonal antibody, collecting the productcontaining an antibody against the erythropoietin receptor, etc. fromthe immunized animal, and then isolating and purifying the antibody.

[0081] As for the complex between an immune antigen and a carrierprotein used for immunizing a mammal, the type of the carrier proteinand the mixing ratio between the carrier and hapten may be any type andin any ratio as long as the antibody can be efficiently produced to thehapten, which is crosslinked with the carrier and used for immunization.For example, bovine serum albumin, bovine thyroglobulin or keyholelimpet hemocyanin is coupled to hapten in a carrier-to-hapten weightratio of about 0.1 to about 20, preferably about 1 to about 5.

[0082] A variety of condensation agents can be used for the coupling ofa carrier to hapten. Glutaraldehyde, carbodiimide, maleimide activatedester and activated ester reagents containing a thiol group or adithiopyridyl group, etc. can be used for the coupling.

[0083] The condensation product is administered alone or together with acarrier or a diluent to a warm-blooded animal at a site capable ofproducing an antibody. In order to enhance the antibody productivity,Freund's complete adjuvant or Freund's incomplete adjuvant may beadministered. The administration is usually carried out once every 2 to6 weeks and 3 to 10 times in total.

[0084] The polyclonal antibody can be collected from the blood, ascites,etc., preferably from the blood of mammals immunized by theabove-mentioned method.

[0085] The polyclonal antibody titer in the antiserum can be determinedby the same procedure as in the serum antibody titer described above.The polyclonal antibody can be isolated and purified according to thesame method for isolation and purification of an antibody as used forthe monoclonal antibody described above.

[0086] Since the erythropoietin receptor antagonist or a salt thereof ora prodrug thereof to be contained in the agent of the present inventionhas an inhibitory effect on tumor proliferation, etc., the agent of thepresent invention is useful as a agent for prophylaxis or treatment ofproliferative organ diseases (e.g., primary, metastatic or recurrenttumors such as breast cancer, prostate cancer, pancreatic cancer,gastric cancer, pulmonary cancer, bowel cancer (colon cancer, rectumcancer, anal cancer), esophageal cancer, duodenal cancer, head and neckcancer (lingual cancer, pharyngeal cancer, larynx cancer), brain tumor,neurilemmoma, non-small-cell pulmonary cancer, small-cell pulmonarycancer, hepatic cancer, renal cancer, bile duct cancer, uterus cancer(uterine body cancer, uterine cervical cancer), ovarian cancer, bladdercancer, skin cancer, angioma, malignant lymphoma, malignant melanoma,thyroid cancer, bone tumor, angioma, angiofibroma, retinal sarcoma,penile cancer, childhood solid cancer, Kaposi's sarcoma, Kaposi'ssarcoma due to AIDS, maxillary sinus tumor, fibrous histiocytoma, smoothmuscle sarcoma, rhabdomyosarcoma, liposarcoma, hysteromyoma,osteoblastoma, osteosarcoma, chondosarcoma, cancerous mesotherioma,leukemia, etc., etc.), chronic arthritic diseases such as rheumatoidarthritis, rheumatoid diseases, chronic arthritis of collagenosis,tendovaginitis, etc., interstitial proliferative disease such ashypertrophic scars, keloid, pulmonary fibrosis, etc. for mammals (e.g.,mouse, rat, hamster, rabbit, cat, dog, bovine, sheep, monkey, human,etc.).

[0087] The agent of the present invention has low toxicity and is safe.

[0088] The erythropoietin receptor antagonist or a salt thereof or aprodrug thereof to be contained in the agent of the present inventionhas low toxicity, and can be safely administered alone or as a mixturewith a pharmaceutically acceptable carrier in the form of apharmaceutical preparation such as tablets (inclusive of sugar-coatedtablets and film-coated tablets), powder, granules, capsules (inclusiveof soft capsules), liquid agents, injection, suppositories orsustained-release agents according to a means known per se generallyused for producing a pharmaceutical preparation, orally or parenterally(e.g., topically, rectally, intravenously, subcutaneously,intramuscularly, intranasally, intravaginally, via oral mucosa, viapulmonary mucosa or via transocular administration, etc.).

[0089] Examples of a pharmaceutically acceptable carrier that may beused for preparation of the agent of the present invention includevarious organic or inorganic carriers that are conventionally used as apharmaceutical material. For example, excipients, lubricants, bindersand disintegrators can be used for solid preparations; and solvents,solubilizing agents, suspending agents, isotonic agents, buffer agents,soothing agents, etc., can be used for liquid preparations. Ifnecessary, conventional additives such as preservatives, antioxidants,coloring agents, sweetening agents, adsorbing agents and wetting agentscan be also used in an appropriate amount.

[0090] Examples of excipients include lactose, white sugar, D-mannitol,starch, corn starch, crystalline cellulose, light anhydrous silicicacid, etc.

[0091] Examples of lubricants include magnesium stearate, calciumstearate, talc, colloidal silica, etc.

[0092] Examples of binders include crystalline cellulose, white sugar,D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinyl pyrrolidone, starch, sucrose, gelatin, methylcellulose, carboxymethyl cellulose sodium, etc.

[0093] Examples of disintegrators include starch,carboxymethylcellulose, carboxymethylcellulose calcium, carboxymethylstarch sodium, L-hydroxypropylcellulose, etc.

[0094] Examples of solvents include water for injection, alcohol,propyleneglycol, Macrogol, sesame oil, corn oil, olive oil, etc.

[0095] Examples of solubilizing agents include polyethylene glycol,propylene glycol, D-mannitol, benzyl benzoate, ethanol,trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodiumcitrate, etc.

[0096] Examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid,lecithin, benzalkonium chloride, benzethonium chloride, glycerinmonostearate, etc.; and hydrophilic polymers such as polyvinylalcohol,polyvinyl pyrrolidone, carboxymethyl cellulose sodium, methyl cellulose,hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropylcellulose, etc.

[0097] Examples of isotonic agents include glucose, D-sorbitol, sodiumchloride, glycerin, D-mannitol, etc.

[0098] Examples of buffer agents include phosphate buffer, acetatebuffer, carbonate buffer, citrate buffer, etc.

[0099] Examples of soothing agents include benzyl alcohol, etc.

[0100] Examples of preservatives include p-hydroxybenzoic esters,chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid,sorbic acid, etc.

[0101] Examples of antioxidants include sulfite, ascorbic acid,α-tocopherol, etc.

[0102] The prepared injection solution is generally filled in a suitableample. When be administered, the above-described composition forinjection can be dissolved in a conventional aqueous diluent to form aliquid agent. Examples of the aqueous diluent include a glucose aqueoussolution, saline, Ringer's solution, nutrition supplemental agentliquid, etc.

[0103] Since the thus obtained pharmaceutical preparation is safe andlow toxic, it can be administered to human or non-human mammals (e.g.,rat, mouse, rabbit, sheep, goat, swine, bovine, horse, cat, dog, monkey,etc.).

[0104] When the injection contains phosphoric acid or a salt thereof,the concentration of sodium phosphate or potassium phosphate in theinjection is about 0.1 mM to 500 mM, preferably about 1 mM to 100 mM.

[0105] Methods of producing a sterile preparation include, but notlimited to, a method in which the whole production steps are carried outaseptically, a gamma-ray sterilization method, a sterilization method byaddition of an antiseptic, etc.

[0106] In the agent of the present invention, the content of theerythropoietin receptor antagonist or a salt thereof or a prodrugthereof varies depending on the form of the agent, and is generallyabout 0.1 to about 100 wt %, preferably about 10 to about 99.9 wt %,more preferably about 20 to about 90 wt % of the total amount of theagent.

[0107] In the agent of the present invention, the content of componentsother than the erythropoietin receptor antagonist or a salt thereof or aprodrug thereof varies depending on the form of the agent, and isgenerally about 10 to about 99.9 wt %, preferably about 20 to about 90wt % of the total amount of the agent.

[0108] The dosage of the agent of the present invention varies dependingon the kind of the erythropoietin receptor antagonist or a salt thereofor a prodrug thereof, the route of the administration, the condition,the age of the patient, etc. For example, in the case wherein the agentis administered parenterally (e.g., intravenously injected, etc.) fortreatment of proliferative organ disease, the daily dosage is about0.005 to about 50 mg, preferably about 0.05 to about 10 mg, morepreferably about 0.2 to about 4 mg per 1 kg body weight of theerythropoietin receptor antagonist and it may be administered in 1 to 3divided doses.

[0109] The agent of the present invention may comprise or may be used incombination with suitable amounts of other pharmaceuticals besides theerythropoietin receptor antagonist or a salt thereof or a prodrugthereof.

[0110] Examples of such a concomitant drug include various anticanceragents that can be used in treatment of proliferative organ diseases.Specific examples thereof include pharmaceuticals having low immuneinhibitory effect such as endocrine therapeutic agents (LH—RH agonistsand antagonists, sex hormone antagonists, sex hormone synthesisinhibitors, etc.), pharmaceuticals targeting for cancer-selective geneproducts (EGF receptor, HER2/erb-2, HER3/erb-3, HER4/erb-4, PDGFreceptor, VEGF receptor, etc.) such as tyrosine kinase, chemotherapeuticagents, and agents for cancer vaccine therapy, etc.

[0111] Examples of agents for cancer vaccine therapy include (1)proteins derived from tumor antigens or similar tumor cells and fragmentpeptides thereof, and fused proteins comprising said proteins orpeptides, (2) DNA fragments encoding said proteins or peptides and beingcapable of expression in vivo, and liposomes comprising said fragments,and (3) viruses or plasmids comprising said DNA fragments.

[0112] Examples of proteins derived from tumor cells which can be usedas an agent for cancer vaccine therapy include melanoma-related antigens(e.g., MAGE-1, MAGE-3, MART-1, gp100, tyrosine kinase, etc.), prostatespecific antigens (PSA), HER2 protein, MUC-1 mucin, hCG, gastrin, heatshock protein, E7 protein of human papilloma virus, carcinoembryonicantigens (CEA), mutated Ras protein, etc.

[0113] Accordingly, the agent of the present invention exhibits superiorprophylactic or therapic effect on proliferative organ diseases even ifused alone. Alternatively, it can be used as a combination with one ortwo kinds of the agent. Furthermore, the effect can be increased byconcomitantly using the agent with one or more of other anticanceragents (concomitant use of multiple drugs). Another advantage of theconcomitant use is that the amount used of the each drug can be reduced,which decreases side effects and remarkably contributes to improvementin Quality of Life for cancer patients (e.g., alleviation of PerformanceStasis and pain, suppression of edema, an increase in apetite, anincrease in body weight, etc.).

[0114] Specific examples of the concomitant agent that can be used withthe agent of the present invention are as follows.

[0115] Examples of the “endocrine therapeutic agents” includefosfestrol, diethylstilbestrol, chlorotrianisene, medroxyprogesteroneacetate, megestrol acetate, chlormadinone acetate, cyproterone acetate,danazol, allylestrenol, gestrinone, mepartricin, raloxifene,ormeloxifene, levormeloxifene, antiesterogens (e.g., tamoxifen citrate,toremifene citrate, etc.), pill preparations, mepitiostane,testololactone, aminoglutethimide, LH—RH agonists (e.g., goserelinacetate, buserelin acetate, leuprorelin acetate, etc.), droloxifene,epitiostanol, ethinylestradiol sulfonate, aromatase inhibitors (e.g.,fadrozole hydrochloride, anastrozole, letrozole, exemestane, vorozole,formestane, etc.), antiandrogens (e.g., flutamide, bicalutamide,nilutamide, etc.), 5α-reductase inhibitors (e.g., finasteride,epristeride, etc.), adrenocorticoids (e.g., dexamethasone, prednisolone,betamethasone, triamcinolone, etc.), androgen synthesis inhibitors(e.g., abiraterone, etc.), retinoids and retinoid metabolism retardants(e.g., liarozole etc.), etc.

[0116] Examples of the “chemotherapeutic agent” include alkylatingagents, antimetabolites, anticancer antibiotics, anticancer agentsderived from plants, etc.

[0117] Examples of the “alkylating agents” include nitrogen mustard,nitrogen mustard N-oxide hydrochloride, chlorambucil, cyclophosphamide,ifosphamide, thiotepa, carboquone, improsulfan tosylate, busulfan,nimustine hydrochloride, mitobronitol, melphalan, dacarbazine,ranimustine, estramustine phosphate sodium, triethylenemelamine,carmustine, lomustine, streptozocin, pipobroman, etoglucid, carboplatin,cisplatin, miboplatin, nedaplatin, oxaliplatin, altretamine,ambamustine, dibrospidium hydrochloride, fotemustine, prednimustine,pumitepa, ribomustine, temozolomide, treosulfan, trofosfamide,zinostatin stimalamer, carboquone, adozelesin, cystemustine, bizelesin,etc.

[0118] Examples of the “antimetabolites” include mercaptopurine,6-mercaptopurine riboside, thioinosine, methotrexate, enocitabin,cytarabine, cytarabine ocfosfate, ancitabine hydrochloride, 5-FU drugs(e.g., fluorouracil, tegafur, UFT, doxifluridine, carmofur,galocitabine, emitefur, etc.), aminopterin, calcium leucovorin, tabloid,butocin, calcium folinate, calcium levofolinate, cladribine, emitefur,fludarabine, gemcitabine, hydroxycarbamide, pentostatin, piritrexim,idoxuridine, mitoguazone, tiazofurin, ambamustine, etc.

[0119] Examples of the “anticancer antibiotics” include actinomycin D,actinomycin C, mitomycin C, chromomycin A3, bleomycin hydrochloride,bleomycin sulfate, peplomycin sulfate, daunorubicin hydrochloride,doxorubicin hydrochloride, aclarubicin hydrochloride, pirarubicinhydrochloride, epirubicin hydrochloride, neocarzinostatin, mithramycin,sarkomycin, carzinophilin, mitotane, zorbicin hydrochloride,mitoxantrone hydrochloride, idarubicin hydrochloride, etc.

[0120] Examples of the “anticancer agents derived from plants” includeetoposide, etoposide phosphate, vinblastine sulfate, vincristinesulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel,vinorelbine, etc.

[0121] Examples of the “immunotherapeutic agents (BRM)” includepicibanil, krestin, schizophyllan, lentinan, ubenimex, interferons,interleukins, macrophage colony stimulating factor, granulocyte colonystimulating factor, lymphotoxin, BCG vaccine, Corynebacterium parvum,levamisole, polysaccharide K, procodazole, etc.

[0122] The “cell growth factor” in the “drug that inhibits the effect ofcell growth factor and receptor thereof” may be any substance as long asit promotes proliferation of cells and generally includes peptideshaving a molecular weight of not more than 20,000 and capable ofexerting the action at a low concentration through binding to areceptor. Specifically, there can be mentioned (1) EGF (epidermal growthfactor) or substances having the substantially the same activity [e.g.,EGF, heregulin (HER2 ligand), etc.], (2) insulin or substances havingsubstantially the same activity [e.g., insulin, IGF (insulin-like growthfactor)-l, IGF-2, etc.], (3) FGF (fibroblast growth factor) orsubstances having substantially the same activity (acidic FGF, basicFGF, KGF (keratinocyte growth factor), FGF-10, etc.), and (4) other cellgrowth factors [e.g., CSF (colony stimulating factor), IL-2(interleukin-2), NGF (nerve growth factor), PDGF (platelet-derivedgrowth factor), TGF-β (transforming growth factor β), HGF (hepatocytegrowth factor), VEGF (vascular endothelial growth factor, etc.), etc.

[0123] The above-mentioned “cell growth factor receptor” may be anyreceptor as long as it can bind to the above-mentioned cell growthfactor, and examples thereof include EGF receptor, heregulin receptor(HER2), insulin receptor-1, insulin receptor-2, IGF receptor, FGFreceptor-1, FGF receptor-2, etc.

[0124] The “drug that inhibits the effect of cell growth factor”includes herceptin (HER2 receptor antibody), etc.

[0125] Besides the above-mentioned drugs, L-asparaginase, aceglatone,procarbazine hydrochloride, protoporphyrin-cobalt complex salt,hematoporphyrin mercury sodium, topoisomerase I inhibitors (e.g.,irinotecan, topotecan, etc.), topoisomerase II inhibitors (e.g.,sobuzoxane, etc.), lyase inhibitors, endothelin antagonists (e.g.,ABT-627, etc.), differentiation inducers (e.g., retinoid, vitamin D,etc.), neovascularization inhibitors, α-blockers (e.g., tamuslosinhydrochloride, etc.), insulin resistance improving agents (e.g.,pioglitazone, rosiglitazone (maleate), GI-262570, JTT-501, MCC-555,YM-440, KRP-297, CS-011, FK-614, compounds described in WO99/58510(e.g.,(E)-4-[4-(5-methyl-2-phenyl-4-oxazolylmethoxy)benzyloxyimino]-4-phenylbutyricacid, etc.), etc.), angiotensin II antagonists (e.g., losartan,eprosartan, candesartan cilexetil, valsartan, telmisartan, irbesartan,tasosartan, olmesartan and active metabolites thereof (e.g.,candesartan, etc.), etc.), cancer antigens, DNA, lectin, glucide, lipid,etc. can also be used.

[0126] Examples of the salts of the concomitant drugs include saltssimilar to the above-mentioned salts of the erythropoietin receptorantagonist.

[0127] For the concomitant use of the agent of the present invention andthe concomitant drug, the administration timing of the agent of thepresent invention and the concomitant drug is not specifically limited.The agent of the present invention and the concomitant drug may beadministered simultaneously or separately with a time lag to a subjectto be administered. The dosage of the concomitant drug may be determinedbased on clinically used dosage, and can be appropriately selecteddepending on the subject to be administered, administration route,disease, combination, etc.

[0128] The dosage forms of the agent of the present invention and theconcomitant drug are not specifically limited as long as the agent ofthe present invention and the concomitant drug are combined when theyare administered. Examples of such dosage form include (1)administration of a single preparation obtained by formulating the agentof the present invention and a concomitant drug together; (2)simultaneous administration of two different preparations obtained byformulating the agent of the present invention and a concomitant drugseparately via the same administration route; (3) separateadministration with a time lag of two different preparations obtained byformulating the agent of the present invention and a concomitant drugseparately via the same administration route; (4) simultaneousadministration of two different preparations obtained by formulating theagent of the present invention and a concomitant drug separately viadifferent administration routes; and 5) separate administration with atime lag of two different preparations obtained by formulating the agentof the present invention and a concomitant drug separately via differentadministration routes (e.g., administration of the agent of the presentinvention followed by administration of a concomitant drug, oradministration in the reverse order). Hereinafter these dosage forms arecollectively abbreviated to the concomitant agent of the presentinvention.

[0129] The concomitant agent of the present invention has low toxicity,and a pharmaceutical composition can be prepared by mixing the agent ofthe present invention and/or the above-mentioned concomitant drug with apharmaceutically acceptable carrier according to a method known per se.

[0130] A pharmaceutically acceptable carrier that may be used forpreparation of the concomitant agent of the present invention may besimilar to those for the above-mentioned pharmaceutical composition ofthe present invention.

[0131] When the agent of the present invention and the concomitant drugare formulated simultaneously and used as a single preparation, thecontent of the erythropoietin receptor antagonist or a salt thereof or aprodrug thereof in the concomitant agent of the present invention variesdepending on the form of the preparation, and is generally about 0.1 toabout 100 wt %, preferably about 10 to about 99.9 wt %, more preferablyabout 20 to about 90 wt % of the total amount of the preparation.

[0132] The content of a concomitant drug in the concomitant agent of thepresent invention also varies depending on the form of the preparation,and is generally about 0.1 to about 100 wt %, preferably about 10 toabout 99.9 wt %, more preferably about 20 to about 90 wt % of the totalamount of the preparation.

[0133] In the concomitant agent of the present invention, the content ofcomponents other than the erythropoietin receptor antagonist or a saltthereof or a prodrug thereof and the concomitant agent varies dependingon the form of the preparation, and is generally about 10 to about 99.9wt %, preferably about 20 to about 90 wt % of the total amount of thepreparation.

[0134] The combination ratio between the erythropoietin receptorantagonist or a salt thereof or a prodrug thereof and a concomitant drugin the concomitant agent of the present invention can be appropriatelyselected depending on a subject to be administrated, route ofadministration, diseases, etc.

[0135] The dosage of the concomitant agent of the present inventionvaries depending on the kind of the erythropoietin receptor antagonistor a salt thereof or a prodrug thereof, the kind of the concomitantdrug, route of administration, condition, age of a patient, etc. Forexample, in the case of oral administration for treatment of cancer, thedaily dosage is about 0.005 to about 50 mg, preferably about 0.05 toabout 10 mg, more preferably about 0.2 to about 4 mg per 1 kg bodyweight of the erythropoietin receptor antagonist or a salt thereof or aprodrug thereof and the concomitant drug and it may be administered in 1to 3 divided doses.

[0136] The same contents may be applied to the case where theerythropoietin receptor antagonist to be contained in the agent of thepresent invention and a concomitant drug are formulated into differentpreparation respectively.

[0137] When the erythropoietin receptor antagonist to be contained inthe agent of the present invention and a concomitant drug are formulatedrespectively into different preparations and subjected to concomitantuse, the agent of the present invention and the pharmaceuticalcomposition comprising a concomitant drug may be administeredsimultaneously. Alternatively, the agent of the present invention may beadministered after the pharmaceutical composition comprising aconcomitant drug is administered, or the pharmaceutical compositioncomprising a concomitant drug may be administered after the agent of thepresent invention is administered. When they are separately administeredwith a time lag, the time lag varies depending on the active componentto be administered, dosage form, and administration method. For example,when the pharmaceutical composition comprising a concomitant drug isadministered first, the agent of the present invention is administeredwithin 1 min to 3 days, preferably 10 min to 1 day, more preferably 15min to 1 hr after the pharmaceutical composition comprising aconcomitant drug is administered. When the agent of the presentinvention is administered first, the pharmaceutical compositioncomprising a concomitant drug is administered within 1 min to 1 day,preferably 10 min to 6 hrs, more preferably 15 min to 1 hr after theagent of the present invention is administered.

[0138] The agent of the present invention can prevent or treat diseasesmore effectively by a combination of the administration alone or theconcomitant administration with a concomitant drug as described above,and 1 to 3 kinds of non-drug therapies. Examples of non-drug therapyinclude operation, radiotherapy, gene therapy, thermotherapy,cryotherapy, burning therapy using laser, etc., and two or more kinds ofthese therapies can be combined.

[0139] For example, the agent of the present invention or theabove-mentioned concomitant agent is used before or after an operation,or is used before or after treatment with a combination of two or threekinds of these therapies, whereby effects such as inhibition ofexpression of resistance, extension of Disease-Free Survival,suppression of metastasis or recurrence of cancer, life lengthening,etc. can be obtained.

[0140] The treatment using the agent of the present invention or theabove-mentioned concomitant agent may also be combined with supportivetherapy [(i) administration of antibiotics (e.g., β-lactams such aspansporin, macrolides such as clarithromycin) against various infectiouscomplications, (ii) administration of high calorie parenteral fluid, anamino acid preparation or a multiple-vitamin preparation for improvingmalnutrition, (iii) administration of morphine for alleviating pain,(iv) administration of an agent for alleviating side effects such asnausea, vomit, loss of apetite, diarrhea, leukopenia, thrombocytopenia,decrease in hemoglobin concentration, alopecia, hepatopathy,nephropathy, DIC, and fever and (v) administration of an agent forsuppressing multiple drug resistance of cancer, etc.].

[0141] Before or after the above-mentioned treatment, the agent of thepresent invention or the above-mentioned concomitant agent is preferablyadministered by oral administration (including sustained-release),intravenous administration (including bolus, infusion, clathrates),subcutaneous or intramuscular injection (inclusive of bolus, infusion,sustained-release), percutaneous, intratumor or proximal administration.

[0142] When the agent of the present invention or the above-mentionedconcomitant agent is administered before an operation etc., it may beadministered once about 30 min to about 24 hr before the operation etc.,or may be administered in one to three cycles from about 3 months toabout 6 months before an operation etc. Such administration of the agentof the present invention or the above-mentioned concomitant agent beforean operation etc. can lead, for example, reduction in the size of cancertissues, whereby the operation etc. can be carried out easily.

[0143] When the pharmaceutical composition of the present invention orthe concomitant agent of the present invention is administered after anoperation etc., it can be administered about 30 min to 24 hr after theoperation etc., for example, repeatedly in the unit of several weeks to3 months. Such administration of the agent of the present invention orthe above-mentioned concomitant agent after an operation etc. canenhance the effect of the operation etc.

[0144] In the present specification and drawings, the codes of bases andamino acids are denoted in accordance with the IUPAC-IUB Commission onBiochemical Nomenclature or by the conventional codes in the art,examples of which are shown below. For amino acids that may have theoptical isomers, L form is presented unless otherwise indicated.

[0145] DNA: deoxyribonucleic acid

[0146] cDNA: complementary deoxyribonucleic acid

[0147] Gly: glycine

[0148] Ala: alanine

[0149] Leu: leucine

[0150] Val: valine

[0151] Pro: proline

[0152] Ser: serine

[0153] Cys: cysteine

[0154] Thr: threonine

[0155] Tyr: tyrosine

[0156] His: histidine

[0157] Trp: tryptophan

[0158] Gln: glutamine

[0159] Lys: lysine

[0160] Phe: phenylalanine

[0161] The sequence identification numbers (SEQ ID NO:) in the sequencelisting of the present specification indicate the following sequence,respectively.

[0162] [SEQ ID NO: 1] indicates the amino acid sequence of EMP9.

[0163] The present invention is described in detail below with referenceto Examples, Reference Examples and Test Examples. However, the scope ofthe present invention should not be construed to be limited to these.

EXAMPLE 1

[0164] 1. Preparation of EMP9

[0165] EMP9 was synthesized by a known method and dissolved in saline.The solution was colored with addition of Evans blue at the finalconcentration of 0.25%.

[0166] 2. Heterotransplantation

[0167] Five-week old nude mice (Balb/c Jcl-nu, Japan Clea Inc.) werepurchased, maintained in a germfree chamber for a week foracclimatization and then used. 5×10⁶ cells of malignant melanoma cellstrains (P39, Utsumi and Elkind, 1993) were suspended in a culturesolution (0.1 ml) and the suspension was injected subcutaneouslyinterscapularly into the mice. After a few weeks, formation of tumor wasobserved on the outer skin surface. The major axis x minor axis x heightof the tumor was measured twice a week. The tumor was observed until itssize became 6×7×7 mm, thereafter administration was started. After theadministration, the size of the tumor was measured in a similar manner.

[0168] 3. Method of Administration

[0169] Four doses of 0.1 ml of a 0.5 mg/ml EMP9 solution wereadministered intraperitoneally at intervals of 1 hr. After 24 hr and 48hr, 0.1 ml of the solution was administered similarly 4 times,respectively. On the 7th day from the first administration, the tumorwas excised.

[0170] 4. Analysis of Change in Tumor Due to Administration

[0171] The excised tumor was fixed in Zamboni fixing liquid or frozen inliquid nitrogen. The tumor fixed in Zamboni fixing liquid was dividedinto several tissue masses. The plural samples collected from therespective masses were embedded to prepare tissue sections thereof. Theproliferating cells were stained using PCNA antibody and the vascularendothelial cells were stained using CD31 antibody, respectively.Hematoxylin was used for nuclear staining. Each of the section specimenswas observed under a microscope at 200-fold magnification. The cellnumber in 20 compartments wherein one compartment has an area of17.6×10⁻³ mm² was counted (the cell number was not less than 4,500). Thecell proliferation ratio was calculated from the following equation andrepresented in [Table 1]. Furthermore, the number of blood vessel in 100compartments was counted, and the average number per a compartment(13.1×10⁻² mm²) was calculated and represented in [Table 1]. Staticprocessing were carried out by Chi-square test for the cellproliferation ratio and by Student's t-test for the number of bloodvessel, and a significant difference was observed between the group inwhich saline was administered and the group in which EMP9 wasadministered (P<0.05).

Proliferation ratio (%)=(number of proliferating cells/number of livingcells)×100

[0172] TABLE 1 Proliferation ratio Number of blood vessel Treatment (%)(average ± standard error) EMP9 3.03  0.64 ± 0.10 Saline 11.23 10.65 ±0.56

[0173] The result of Table 1 shows that the EMP9 has superior inhibitoryeffects on both tumor proliferation and neovascular proliferation.

EXAMPLE 2

[0174] (1) EMP9: 10.0 g

[0175] (2) lactose: 60.0 g

[0176] (3) corn starch: 35.0 g

[0177] (4) gelatin: 3.0 g

[0178] (5) magnesium stearate: 2.0 g

[0179] A mixture of EMP9 (10.0 g), lactose (60.0 g) and corn starch(35.0 g) was granulated by using 10 wt % aqueous gelatin solution (30ml, 3.0 g as gelatin) and passing a sieve having a mesh size of 1 mm,and thereafter, the granules were dried at 40° C. and sieved again. Theobtained granules were mixed with magnesium stearate (2.0 g) and themixture was compressed. The obtained core tablets were coated with sugarcoating consisting of an aqueous suspension of sucrose, titaniumdioxide, talc and gum arabic. The coated tablets were glazed withbeeswax to give coated tablets (1000 tablets).

EXAMPLE 3

[0180] (1) EMP9: 10.0 g

[0181] (2) lactose: 70.0 g

[0182] (3) corn starch: 50.0 g

[0183] (4) soluble starch: 7.0 g

[0184] (5) magnesium stearate: 3.0 g

[0185] EMP9 (10.0 g) and magnesium stearate (3.0 g) were granulatedusing an aqueous solution of a soluble starch (70 ml, 7.0 g as solublestarch). The granules were dried and mixed with lactose (70.0 g) andcorn starch (50.0 g). The mixture was compressed to give tablets (1000tablets).

REFERENCE EXAMPLE 1

[0186] (1) leuprorelin acetate: 10.0 mg

[0187] (2) lactose: 70.0 mg

[0188] (3) corn starch: 50.0 mg

[0189] (4) solubilized starch: 7.0 mg

[0190] (5) magnesium stearate: 3.0 mg

[0191] Leuprorelin acetate (10.0 mg) and magnesium stearate (3.0 mg) aregranulated using an aqueous solution of a solubilized starch (0.07 ml,7.0 mg as solubilized starch), and then the granules are dried and mixedwith lactose (70.0 mg) and cornstarch (50.0 mg). The mixture iscompressed to give tablets.

PREPARATION EXAMPLE 1

[0192] The preparation obtained in Example 1 or 2 and the preparationobtained in Reference Example 1 are combined.

[0193] Industrial Applicability:

[0194] An erythropoietin receptor antagonist or a prodrug thereof hassuperior inhibitory effects on both tumor proliferation and neovascularproliferation, and is useful as an agent for prophylaxis or treatment ofproliferative organ diseases, chronic arthritic diseases, hypertrophicscars or keloid. Specifically, it is effective against solid tumors withvascular proliferation.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 1 <210> SEQ ID NO 1 <211>LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Human <300> PUBLICATIONINFORMATION: <301> AUTHORS: Dana L. Johnson et al <302> TITLE:Identification of a 13 Amino Acid Peptide Mimetic of Erythropoietin andDescription of Amino Acids Critical for the Mimetic Activity of EMP1<303> JOURNAL: Biochemistry <304> VOLUME: 37 <305> ISSUE: 11 <306>PAGES: 3699-3710 <400> SEQUENCE: 1 Gly Gly Thr Tyr Ser Cys His Phe AlaPro Leu Thr Trp Val Cys 1 5 10 15 Lys Pro Gln Gly Gly 20

1. an agent for prophylaxis or treatment of proliferative organdiseases, chronic arthritic diseases, hypertrophic scars or keloid,comprising an erythropoietin receptor antagonist or a salt thereof: 2.The agent according to claim 1, wherein the erythropoietin receptorantagonist or a salt thereof is a low molecular weight erythropoietinreceptor antagonist or a salt thereof.
 3. The agent according to claim1, wherein the erythropoietin receptor antagonist or a salt thereof is alow molecular weight peptidic erythropoietin receptor antagonist or asalt thereof.
 4. The agent according to claim 1, wherein theerythropoietin receptor antagonist or a salt thereof is anerythropoietin mimetic peptide or a salt thereof.
 5. The agent accordingto claim 1, wherein the erythropoietin receptor antagonist or a saltthereof is erythropoietin mimetic peptide 9 or a salt thereof.
 6. Theagent according to claim 1, wherein the erythropoietin receptorantagonist or a salt thereof is a peptide comprising an amino acidsequence identical or substantially identical with the amino acidsequence of SEQ ID NO: 1 or a salt thereof.
 7. The agent according toclaim 1, wherein the erythropoietin receptor antagonist or a saltthereof is an anti-erythropoietin receptor antibody or a salt thereof.8. The agent according to claim 1, wherein the proliferative organdisease is cancer or tumor.
 9. The agent according to claim 1, whereinthe chronic arthritic disease is rheumatoid arthritis, rheumatoiddiseases, chronic arthritis of collagenosis or tendovaginitis.
 10. Avascular proliferation suppressing agent comprising an erythropoietinreceptor antagonist or a salt thereof.
 11. A method for prophylaxis ortreatment of proliferative organ diseases, chronic arthritic diseases,hypertrophic scars or keloid, comprising administering an effectiveamount of an erythropoietin receptor antagonist or a salt thereof to amammal.
 12. A method for prophylaxis or treatment of proliferative organdiseases, comprising administering an effective amount of anerythropoietin receptor antagonist or a salt thereof in combination withan effective amount of another anticancer agent or a salt thereof to amammal.
 13. (Cancelled)